One of the major non-infectious risks from blood transfusions is the development of transfusion-related acute lung injury (TRALI), which is the number one cause of transfusion-related mortality in the United States. We have previously shown in a mouse model of TRALI that neutrophils and platelets are both required to produce acute lung endothelial injury, protein permeability, and pulmonary edema. However, the mechanisms by which neutrophils and platelets potentially interact and ultimately lead to lung injury are not known. In this application, we will test potential mechanisms in three scientific aims. In Aim 1, we will test for the presence of neutrophil-platelet aggregates in TRALI and determine the molecular mechanisms responsible for the aggregates by using bone marrow chimeras and in vitro studies with cells isolated from MHC Class I-null mice and Fcg receptor knockouts. We will also test pharmacologic inhibitors of platelets and the mechanisms by which these inhibitors may ameliorate lung injury. In Aim 2, we will determine how neutrophils lead to lung endothelial injury by using mice with loss of function mutations in neutrophil proteases and ROS production. We hypothesize that neutrophil extracellular traps (NETs) will be formed in TRALI in a platelet-dependent process that exposes extracellular histones leading to lung endothelial injury. In Aim 3, we will use a new application of two-photon microscopy in the live, mouse lung to image the temporal sequence of neutrophil and platelet recruitment in our mouse model of TRALI. Using intravital microscopy, we will also determine the spatial relationships between neutrophils and platelets in the injured lung and how this influences NET formation in the lung microvasculature. The results from this investigation will elucidate the mechanisms of lung injury in TRALI, and by focusing on the roles of platelet activation and NET formation it may be possible to identify novel pharmacologic approaches to treating acute lung injury. PUBLIC HEALTH RELEVANCE: Blood transfusions may acutely injure the lungs in a process termed transfusion-related acute lung injury (TRALI). Using a mouse model of TRALI, we have discovered that neutrophils and platelets are responsible for this injury. In this proposal, we will determine how neutrophils and platelets interact with each other in the lung microcirculation to ultimately produce lung injury.